An Automated Nanowell-Array Workflow for Quantitative Multiplexed Single-Cell Proteomics Sample Preparation at High Sensitivity.

Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation including multiplexed labeling up to 16-plex with high sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE, reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1500 protein groups per TMT10-plex with reduced or eliminated need for a carrier proteome. We evaluated the effect of wider precursor isolation windows at single-cell input levels and found that using 2 Da isolation windows increased overall sensitivity without significantly impacting interference. Using the dedicated mass spectrometry acquisition strategies detailed here, we identified on average close to 2000 proteins per TMT10-plex across 170 multiplexed single cells that readily distinguished human cell types. Overall, our workflow combines highly efficient sample preparation, chromatographic and ion mobility-based filtering, rapid wide-window data-dependent acquisition analysis, and intelligent data analysis for optimal multiplexed single-cell proteomics. This versatile and automated proteoCHIP-based sample preparation approach is sufficiently sensitive to drive biological applications of single-cell proteomics and can be readily adopted by proteomics laboratories.

Arraying of Single Cells for Quantitative High Throughput Laser Ablation ICP-TOF-MS.

Arraying of single cells for mass spectrometric analysis is a considerable bioanalytical challenge. In this study, we employ a novel single cell arraying technology for quantitative analysis and isotopic fingerprinting by laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS). The single cell arraying approach is based on a piezo-acoustic microarrayer with software for automated optical detection of cells within the piezo dispense capillary (PDC) prior to arraying. Using optimized parameters, single cell occupancy of >99%, high throughput (up to 550 cells per hour), and a high cell recovery of >66% is achieved. LA-ICP-TOF-MS is employed to detect naturally occurring isotopes in the whole mass range as fingerprints of individual cells. Moreover, precise quantitative determination of metal-containing cell dyes is possible down to contents of ∼100 ag using calibration standards which were produced using the same arrayer.

A high-throughput polymer microarray approach for identifying defined substrates for mesenchymal stem cells.

Mesenchymal stem cells (MSCs) hold great promise in regenerative medicine due to their wide multilineage potential as well as their ability to suppress/modulate the immune response. Maintaining these cells in vitro and expanding them on a clinically relevant scale remains a challenge that needs to be addressed to realise their full potential. Current culture methods for MSCs typically rely on animal sourced substrates and often result in a heterogeneous population of cells with varying degrees of differentiation capacity. Here, a high-throughput platform was used to identify synthetic substrates for MSC culture that not only facilitated growth but also maintained the MSC phenotype. Two polymers, PU157 (synthesised from poly(butyleneglycol) and 4,4'-methylenediphenyldiisocyanate with 3-(dimethylamino)-1,2-propanediol as a chain extender) and PA338 (N-methylaniline modified poly(methylmethacrylate-co-glycidylmethacrylate)) were able to maintain the growth and phenotype of human embryonic derived mesenchymal progenitors (hES-MPs) and adipose derived MSCs (ADMSCs) for five and ten passages, respectively. Cell phenotype and multipotency were confirmed by flow cytometry analysis of ten MSC markers and differentiation analysis. These new polymer substrates provide a chemically defined synthetic surface for efficient, long-term MSC culture.

A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells.

Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth, while mechanical, enzymatic or chemical cell dissociation methods are used for cellular passaging. However, these methods are ill defined, thus introducing variability into the system, and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate, which support long-term human embryonic stem cell growth and pluripotency over a period of 2-6 months. The hydrogels permitted gentle, reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used, undefined biological substrates represent a flexible and scalable approach for improving the definition, efficacy and safety of human embryonic stem cell culture systems for research, industrial and clinical applications.

Fingerprinting polymer microarrays.

The incubation of "polymer microarrays" with labelled proteins and carbohydrates demonstrated polymer selective binding, giving an approach to cellular fingerprinting and offering a possible alternative to current arraying platforms for partitioning and analysis of complex cellular components.

A microarray approach to the identification of polyurethanes for the isolation of human skeletal progenitor cells and augmentation of skeletal cell growth.

The present study has examined the efficacy of a polymer microarray platform to screen a library of polyurethanes for applications such as human skeletal progenitor cell isolation and surface modification of tissue engineering scaffolds to enhance skeletal cell growth and differentiation. Analysis of polyurethane microarrays incubated with adult human bone marrow-derived STRO-1+ skeletal progenitor cells identified 31 polyurethanes (from the entire library of 120 polyurethanes) capable of binding to the STRO-1+ cells. Four polyurethanes (out of the 31 identified in the previous screen) were able to selectively immobilise cells of the STRO-1+ fraction from the heterogeneous human bone marrow mononuclear cell population. These four polyurethanes were highly selective for the STRO-1+ fraction of human bone marrow as they failed to bind STRO-1+ immature osteoblast-like MG63 cells, the STRO-1+ fraction of human fetal skeletal cells and differentiated osteoblast-like SaOs cells. Culture of human bone marrow-derived STRO-1+ cells on fibres of Polyglycolic acid (PGA) fleece surface modified by polyurethane adsorption, in osteogenic conditions, enhanced the expression of early osteogenic genes. Similarly, surface modification of PGA fleece fibres by polyurethane adsorption increased the responsiveness of MG63 cells, cultured on this scaffold, to 1,25 dihydroxy Vitamin D3, as demonstrated by enhanced Osteocalcin expression.

Measurement of glucose in blood with a phenylboronic acid optical sensor.

BACKGROUND: Current methods of glucose monitoring rely predominantly on enzymes such as glucose oxidase for detection. Phenylboronic acid receptors have been proposed as alternative glucose binders. A unique property of these molecules is their ability to bind glucose in a fully reversible covalent manner that facilitates direct continuous measurements. We examined (1) the ability of a phenylboronic-based sensor to measure glucose in blood and blood plasma and (2) the effect on measurement accuracy of a range of potential interferents. We also showed that the sensor is able to track glucose fluctuations occurring at rates mimicking those experienced in vivo. METHOD: In vitro static measurements of glucose in blood and blood plasma were conducted using holographic sensors containing acrylamide, N,N'-methylenebisacrylamide, 3-acrylamidophenylboronic acid, and (3-acrylamidopropyl) trimethylammonium chloride. The same sensors were also used for in vitro measurements performed under flow conditions. RESULTS: The opacity of the liquid had no affect on the ability of the optical sensor to measure glucose in blood or blood plasma. The presence of common antibiotics, diabetic drugs, pain killers, and endogenous substances did not affect the measurement accuracy, as shown by error grid analysis. Ex vivo flow experiments showed that the sensor is able to track changes accurately in concentration occurring in real time without lag or evidence of hysteresis. CONCLUSIONS: The ability of phenylboronic acid sensors to measure glucose in whole blood was demonstrated for the first time. Holographic sensors are ideally suited to continuous blood glucose measurements, being physically and chemically robust and potentially calibration free.

Continuous blood glucose monitoring with a thin-film optical sensor.

BACKGROUND: We recently described a holographic optical sensor with improved selectivity for glucose over fructose that was based on a thin-film polymer hydrogel containing phenylboronic acid receptors. The aim of the present work was to measure glucose in human blood plasma as opposed to simple buffers and track changes in concentration at a rate mimicking glucose changes in vivo. METHODS: We used holographic sensors containing acrylamide, N,N'-methylenebisacrylamide, 3-acrylamidophenylboronic acid, and (3-acrylamidopropyl)trimethylammonium chloride to measure 7 human blood plasma samples at different glucose concentrations (3-33 mmol/L) in static mode. Separately, using a flow cell, the glucose concentration was varied at approximately 0.17-0.28 mmol(-1) x L(-1) x min(-1), and the sensor's ability to continuously monitor glucose was investigated over an extended period. RESULTS: We subjected the results of the ex vivo static measurements to error grid analysis. Of 46 measurements, 42 (91.3%) fell in zone A of a Clarke error grid, and the remainder (8.7%) fell in zone B. The ex vivo flow experiments showed that the sensor is able to accurately track changes in concentration occurring in real time without lag or evidence of hysteresis. CONCLUSIONS: We demonstrate the ability of a phenylboronic acid-based sensor to measure glucose in human blood plasma for the 1st time in vitro. Holographic glucose sensors can be used without recourse to recalibration. Their robust nature, coupled with their format flexibility, makes them an attractive alternative to conventional electrochemical enzyme-based methods of glucose monitoring for people with diabetes.

Microarray platforms for enzymatic and cell-based assays.

This tutorial review introduces the uninitiated to the world of microarrays (or so-called chips) and covers a number of basic concepts such as substrates and surfaces, printing and analysis. It then moves on to look at some newer applications of microarray technology, which include enzyme analysis (notably kinases and proteases) as well as the growing enchantment with so-called cell-based microarrays that offer a unique approach to high-throughput cellular analysis. Finally, it looks forwards and highlights future possible trends and directions in the microarray arena.

Polymer microarrays: identification of substrates for phagocytosis assays.

A polymer microarray of 120 polyurethanes was used to identify polymers that promoted the adhesion of bone marrow dendritic cells (BMDC). Identified polymers were coated onto glass cover slips and shown to be efficient substrates for the immobilisation of these primary cells, which underwent efficient phagocytosis while still presumably maintaining their immature state.

Polymer microarrays for cellular adhesion.

Microarray screening of polymer libraries for cellular adhesion was developed utilising a thin film of agarose to allow unsurpassed localisation of cell binding onto the array substrate and the discovery of cell specific polymers.